Mammalian mitogen-activated protein (MAP) kinases are serine/threonine kinases that mediate intracellular signal transduction pathways (Cobb and Goldsmith, J. Biol. Chem., 270, 14843 (1995); Davis, Mol. Reprod. Dev., 42, 459 (1995)). Members of the MAP kinase family share sequence similarity and conserved structural domains, and include the ERK2 (extracellular signal regulated kinase), JNK (Jun N-terminal kinase), and p38 kinases. JNKs and p38 kinases are activated in response to the pro-inflammatory cytokines TNF-alpha and interleukin-1, and by cellular stress such as heat shock, hyperosmolarity, ultraviolet radiation, lipopolysaccharides and inhibitors of protein synthesis (Derijard et al., Cell, 76, 1025 (1994); Han et al., Science, 265, 808 (1994); Raingeaud et al., J. Biol. Chem., 270, 7420 (1995); Shapiro and Dinarello, Proc. Natl. Acad. Sci. USA, 92, 12230 (1995)). In contrast, ERKs are activated by mitogens and growth factors (Bokemeyer et al., Kidney Int., 49, 1187 (1996)).
ERK2 is a widely distributed protein kinase that achieves maximum activity when both Thr183 and Tyr185 are phosphorylated by the upstream MAP kinase kinase, MEK1 (Anderson et al., Nature, 343, 651 (1990); Crews et al., Science, 258, 478 (1992)). Upon activation, ERK2 phosphorylates many regulatory proteins, including the protein kinases Rsk90 (Bjorbaek et al., J. Biol. Chem., 270, 18848 (1995)) and MAPKAP2 (Rouse et al., Cell, 78, 1027 (1994)), and transcription factors such as ATF2 (Raingeaud et al., Mol. Cell Biol., 16, 1247 (1996)), Elk-1 (Raingeaud et al. 1996), c-Fos (Chen et al., Proc. Natl. Acad. Sci. USA, 90, 10952 (1993)), and c-Myc (Oliver et al., Proc. Soc. Exp. Biol. Med., 210, 162 (1995)). ERK2 is also a downstream target of the Ras/Raf dependent pathways (Moodie et al., Science, 260, 1658 (1993)) and relays the signals from these potentially oncogenic proteins. ERK2 has been shown to play a role in the negative growth control of breast cancer cells (Frey and Mulder, Cancer Res., 57, 628 (1997)) and hyperexpression of ERK2 in human breast cancer has been reported (Sivaraman et al., J Clin. Invest., 99, 1478 (1997)). Activated ERK2 is also implicated in the proliferation of endothelin-stimulated airway smooth muscle cells, associating this kinase with asthma (Whelchel et al., Am. J. Respir. Cell Mol. Biol., 16, 589 (1997)).
Aurora-2 is a serine/threonine protein kinase that is implicated in human cancer, such as colon, breast and other solid tumors. This kinase is involved in protein phosphorylation events that regulate the cell cycle. Specifically, Aurora-2 plays a role in controlling the accurate segregation of chromosomes during mitosis. Misregulation of the cell cycle can lead to cellular proliferation and other abnormalities. In human colon cancer tissue, the aurora-2 protein is overexpressed. See Bischoff et al., EMBO J., 17, 3052-3065 (1998); Schumacher et al., J. Cell Biol., 143, 1635-1646 (1998); Kimura et al., J. Biol. Chem., 272, 13766-13771 (1997).
Glycogen synthase kinase-3 (GSK-3) is a serine/threonine protein kinase comprised of α and β isoforms that are each encoded by distinct genes (Coghlan et al., Chemistry & Biology, 7, 793-803 (2000); Kim and Kimmel, Curr. Opinion Genetics Dev., 10, 508-514 (2000)). GSK-3 has been implicated in various diseases including diabetes, Alzheimer's disease, CNS disorders such as manic depressive disorder and neurodegenerative diseases, and cardiomyocete hypertrophy (WO 99/65897; WO 00/38675; and Haq et al., J. Cell Biol., 151, 117 (2000)). These diseases are caused by, or result in, the abnormal operation of certain cell signaling pathways in which GSK-3 plays a role. GSK-3 phosphorylates and modulates the activity of a number of regulatory proteins. These proteins include glycogen synthase which is the rate limiting enzyme necessary for glycogen synthesis, the microtubule associated protein Tau, the gene transcription factor β-catenin, the translation initiation factor e1F2B, as well as ATP citrate lyase, axin, heat shock factor-1, c-Jun, c-Myc, c-Myb, CREB, and CEPBα. These diverse protein targets implicate GSK-3 in many aspects of cellular metabolism, proliferation, differentiation and development.
In a GSK-3 mediated pathway that is relevant for the treatment of type II diabetes, insulin-induced signaling leads to cellular glucose uptake and glycogen synthesis. Along this pathway, GSK-3 is a negative regulator of the insulin-induced signal. Normally, the presence of insulin causes inhibition of GSK-3 mediated phosphorylation and deactivation of glycogen synthase. The inhibition of GSK-3 leads to increased glycogen synthesis and glucose uptake (Klein et al., PNAS, 93, 8455-9 (1996); Cross et al., Biochem. J., 303, 21-26 (1994); Cohen, Biochem. Soc. Trans., 21, 555-567 (1993); and Massillon et al., Biochem J., 299, 123-128 (1994)). However, in a diabetic patient where the insulin response is impaired, glycogen synthesis and glucose uptake fail to increase despite the presence of relatively high blood levels of insulin. This leads to abnormally high blood levels of glucose with acute and long term effects that may ultimately result in cardiovascular disease, renal failure and blindness. In such patients, the normal insulin-induced inhibition of GSK-3 fails to occur. It has also been reported that in patients with type II diabetes, GSK-3 is overexpressed (WO 00/38675). Therapeutic inhibitors of GSK-3 therefore are considered to be useful for treating diabetic patients suffering from an impaired response to insulin.
GSK-3 activity is associated with Alzheimer's disease. This disease is characterized by the well-known β-amyloid peptide and the formation of intracellular neurofibrillary tangles. The neurofibrillary tangles contain hyperphosphorylated Tau protein where Tau is phosphorylated on abnormal sites. GSK-3 phosphorylates these abnormal sites in cell and animal models. Furthermore, inhibition of GSK-3 prevents hyperphosphorylation of Tau in cells (Lovestone et al., Current Biology, 4, 1077-86 (1994); Brownlees et al., Neuroreport, 8, 3251-55 (1997)). Therefore, GSK-3 activity promotes generation of the neurofibrillary tangles and the progression of Alzheimer's disease.
Another substrate of GSK-3 is β-catenin which is degradated after phosphorylation by GSK-3. Reduced levels of β-catenin have been reported in schizophrenic patients and have also been associated with other diseases related to increase in neuronal cell death (Zhong et al., Nature, 395, 698-702 (1998); Takashima et al., PNAS, 90, 7789-93 (1993); Pei et al., J. Neuropathol. Exp, 56, 70-78 (1997)).
AKT (also known as PKB or Rac-PK beta), a serine/threonine protein kinase, has been shown to be overexpressed in several types of cancer and is a mediator of normal cell functions (Khwaja, A., Nature, 401, 33-34 (1999); Yuan, Z. Q., et al., Oncogene, 19, 2324-2330 (2000); Namikawa, K., et al., J Neurosci., 20, 2875-2886 (2000)). AKT comprises an N-terminal pleckstrin homology (PH) domain, a kinase domain and a C-terminal “tail” region. Three isoforms of human AKT kinase (AKT-1, -2 and -3) have been reported so far (Cheng, J. Q., Proc. Natl. Acad. Sci. USA, 89, 9267-9271 (1992); Brodbeck, D. et al., J. Biol. Chem., 274, 9133-9136 (1999). The PH domain binds 3-phosphoinositides, which are synthesized by phosphatidyl inositol 3-kinase (PI3K) upon stimulation by growth factors such as platelet derived growth factor (PDGF), nerve growth factor (NGF) and insulin-like growth factor (IGF-1) (Kulik et al., Mol. Cell. Biol., 17, 1595-1606 (1997); Hemmings, B. A., Science, 275, 628-630 (1997). Lipid binding to the PH domain promotes translocation of AKT to the plasma membrane and facilitates phosphorylation by another PH-domain-containing protein kinases, PDK1 at Thr308, Thr309, and Thr305 for the AKT isoforms 1, 2 and 3, respectively. A second, as of yet unknown, kinase is required for the phosphorylation of Ser473, Ser474 or Ser472 in the C-terminal tails of AKT-1, -2 and -3 respectively, in order to yield a fully activated AKT enzyme.
Once localized to the membrane, AKT mediates several functions within the cell including the metabolic effects of insulin (Calera, M. R. et al., J. Biol. Chem., 273, 7201-7204 (1998)) induction of differentiation and/or proliferation, protein synthesisans stress responses (Alessi, D. R. et al., Curr. Opin. Genet. Dev., 8, 55-62 (1998)).
Manifestations of altered AKT regulation appear in both injury and disease, the most important role being in cancer. The first account of AKT was in association with human ovarian carcinomas where expression of AKT was found to be amplified in 15% of cases (Cheng, J. Q. et al., Proc. Natl. Acad. Sci. U.S.A., 89, 9267-9271 (1992)). It has also been found to be overexpressed in 12% of pancreatic cancers (Cheng, J. Q. et al., Proc. Natl. Acad. Sci. U.S.A., 93, 3636-3641 (1996)). It was demonstrated that AKT-2 was over-expressed in 12% of ovarian carcinomas and that amplification of AKT was especially frequent in 50% of undifferentiated tumours, suggesting that AKT may also be associated with tumour aggressiveness (Bellacosa, et al., Int. J. Cancer, 64, 280-285 (1995)). Cyclin-dependent kinases (CDKs) are serine/threonine protein kinases consisting of a β-sheet rich amino-terminal lobe and a larger carboxy-terminal lobe that is largely α-helical. The CDKs display the 11 subdomains shared by all protein kinases and range in molecular mass from 33 to 44 kD. This family of kinases, which includes CDK1, CKD2, CDK4, and CDK6, requires phosphorylation at the residue corresponding to CDK2 Thr160 in order to be fully active (Meijer, L., Drug Resistance Updates, 3, 83-88 (2000)).
Each CDK complex is formed from a regulatory cyclin subunit (e.g., cyclin A, B1, B2, D1, D2, D3, and E) and a catalytic kinase subunit (e.g., CDK1, CDK2, CDK4, CDK5, and CDK6). Each different kinase/cyclin pair functions to regulate the different and specific phases of the cell cycle known as the G1, S, G2, and M phases (Nigg, E., Nature Reviews, 2, 21-32 (2001); Flatt, P., Pietenpol, J., Drug Metabolism Reviewvs, 32, 283-305 (2000)).
The CDKs have been implicated in cell proliferation disorders, particularly in cancer. Cell proliferation is a result of the direct or indirect deregulation of the cell division cycle and the CDKs play a critical role in the regulation of the various phases of this cycle. For example, the over-expression of cyclin D1 is commonly associated with numerous human cancers including breast, colon, hepatocellular carcinomas and gliomas (Flatt, P., Pietenpol, J., Drug Metabolism Reviews, 32, 283-305 (2000)). The CDK2/cyclin E complex plays a key role in the progression from the early G1 to S phases of the cell cycle and the overexpression of cyclin E has been associated with various solid tumors. Therefore, inhibitors of cyclins D1, E, or their associated CDKs are useful targets for cancer therapy (Kaubisch, A., Schwartz, G., The Cancer Journal, 6, 192-212 (2000)).
CDKs, especially CDK2, also play a role in apoptosis and T-cell development. CDK2 has been identified as a key regulator of thymocyte apoptosis (Williams, O. et al., European Journal of Immunology, 709-713 (2000)). Stimulation of CDK2 kinase activity is associated with the progression of apoptosis in thymocytes, in response to specific stimuli. Inhibition of CDK2 kinase activity blocks this apoptosis resulting in the protection of thymocytes.
In addition to regulating the cell cycle and apoptosis, the CDKs are directly involved in the process of transcription. Numerous viruses require CDKs for their replication process. Examples where CDK inhibitors restrain viral replication include human cytomegalovirus, herpes virus, and varicella-zoster virus (Meijer, L., Drug Resistance Updates, 3, 83-88 (2000)).
Inhibition of CDK is also useful for the treatment of neurodegenerative disorders such as Alzheimer's disease. The appearance of Paired Helical Filaments (PHF), associated with Alzheimer's disease, is caused by the hyperphosphorylation of Tau protein by CDK5/p25 (Meijer, L., Drug Resistance Updates, 3, 83-88 (2000)).
Another kinase family of interest is Rho-associated coiled-coil forming protein serine/threonine kinase (ROCK), which is believed to be an effector of Ras-related small GTPase Rho. The ROCK family includes p160ROCK (ROCK-1) (Ishizaki et al., EMBO J., 15, 1885-1893 (1996)) and ROKα/Rho-kinase/ROCK-II (Leung et al., J. Biol. Chem., 270, 29051-29054 (1995); Matsui et al., EMBO J., 15, 2208-2216 (1996); Nakagawa et al., FEBS Lett., 392, 189-193 (1996)), protein kinase PKN (Amano et al., Science, 271, 648-650 (1996); Watanabe et al., Science, 271, 645-648 (1996)), and citron and citron kinase (Madaule et al. Nature, 394, 491-494 (1998); Madaule et al., FEBS Lett., 377, 243-248 (1995)). The ROCK family of kinases have been shown to be involved in a variety of functions including Rho-induced formation of actin stress fibers and focal adhesions (Leung et al., Mol. Cell Biol., 16, 5313-5327 (1996); Amano et al., Science, 275, 1308-1311 (1997); Ishizaki et al., FEBS Lett., 404, 118-124 (1997)) and in downregulation of myosin phosphatase (Kimura et al., Science, 273, 245-248 (1996)), platelet activation (Klages et al., J. Cell. Biol., 144, 745-754 (1999)), aortic smooth muscle contraction by various stimuli (Fu et al., FEBS Lett., 440, 183-187 (1998)), thrombin-induced responses of aortic smooth muscle cells (Seasholtz et al., Cir. Res., 84, 1186-1193 (1999)), hypertrophy of cardiomyocytes (Kuwahara et al., FEBS Lett., 452, 314-318 (1999)), bronchial smooth muscle contraction (Yoshii et al., Am. J. Respir. Cell Mol. Biol., 20, 1190-1200 (1999)), smooth muscle contraction and cytoskeletal reorganization of non-muscle cells (Fukata et al., Trends in Pharm. Sci, 22, 32-39 (2001)), activation of volume-regulated anion channels (Nilius et al., J. Physiol., 516, 67-74 (1999)), neurite retraction (Hirose et al., J. Cell. Biol., 141, 1625-1636 (1998)), neutrophil chemotaxis (Niggli, FEBS Lett., 445, 69-72 (1999)), wound healing (Nobes and Hall, J. Cell. Biol., 144, 1235-1244 (1999)), tumor invasion (Itoh et al., Nat. Med., 5, 221-225 (1999)) and cell transformation (Sahai et al., Curr. Biol., 9, 136-145 (1999)). Accordingly, the development of inhibitors of ROCK kinase would be useful as therapeutic agents for the treatment of disorders mediated by the ROCK kinase pathway.
There is a high unmet medical need to develop new therapeutic treatments that are useful in treating or preventing the various conditions associated with ERK2, GSK3, Aurora2, CDK2, AKT3, and ROCK activation. For many of these conditions the currently available treatment options are inadequate.
Accordingly, there is great interest in new and effective inhibitors of protein kinase, including ERK2, GSK3, Aurora2, CDK2, AKT3, and ROCK inhibitors, that are useful in treating or preventing various conditions associated with protein kinase activation.